Early differentiating neurons in the zebrafish are generated in discrete neurogenic domains of the neural plate. The mechanisms by which these discrete domains are defined in zebrafish provide insight about how neurogenesis is regulated in the vertebrate nervous system. Previously we described the role of Zic genes in defining boundaries in the neural plate adjacent to which neurons differentiate. Now we have characterized a fish-specific gene, Zic6, which has been lost in frogs, bird and mammals. Its analysis serves as a basis for understanding the evolution of the Zic genes and the significance of paired arrangement of Zic genes in the genome. [unreadable] The Zic gene family encodes a group of C2H2 zinc-finger transcription factors that are important regulators of early vertebrate development. They are part of a larger Gli/Zic/NKL gene superfamily and, together with the Gli genes, are thought to help define tissue compartments with specific fate within the developing embryo. The Zic genes are typically expressed in ectodermal tissues contributing to the nervous system and neural crest, as well as somatic mesoderm. There is strong experimental support for a combined role of the Zic genes in neurulation, neurogenesis, neural crest specification, and establishment of leftright asymmetry. Deficits in Zic gene family members have been linked to developmental defects such as spina-bifida, holoprosencephaly, and X-linked heterotaxia. Understanding the significance of Zic gene function during embryonic development is confounded by their broadly overlapping expression with the potential for competition for DNA-binding, sites as well as crossregulatory and physical interactions among orthologues. It is therefore essential to define the combined expression of the Zic gene family members and understand their evolutionary relationships. Although there is significant conservation in the structure of the Zic protein DNA-binding domain, consisting of five zinc-fingers, there is also considerable divergence in other parts of the protein that may be correlated with altered post-translational regulation, proteinprotein interactions and repressor/activator activities. The evolutionary diversification among family members may, however, be constrained by their physical arrangement as paired genes (bigenes) that share a limited amount of upstream DNA. [unreadable] Four known vertebrate homologues occur as zic1/zic4 and zic2/zic5 bigenes, with the exception being zic3, which is a single-gene locus, located on the X chromosome in mammals. We have described the structure, genomic context, and embryonic expression of zebrafish zic6 and use this analysis to infer the evolutionary relationships of the Zic family members in vertebrates that include fish, frogs, birds and mammals. The zic6 gene was found to be teleost-specific, occurring among a broad range of fishes, but absent from the genomes of frogs, birds, and mammals. Genomic analysis established that zic6 is paired with zic3, in opposite orientation, as is the case with the zic1/zic4 and zic2a/zic5 gene pairs. Synteny of flanking genes confirmed that the zic3 loci of fish and the other vertebrate taxa are true homologues, supporting the conclusion that zic6 was the product of a chromosomal duplication before the divergence of fishes and tetrapods and was subsequently lost in the tetrapod lineage. The expression of zic6 in the neural plate lacked the lateral and rostral domains typical of the other Zic gene orthologues, indicating it has a different regulatory role during early embryonic development of fish. We are currently examining if interactions between bigenes influences the spatiotemporal pattern of their expression.[unreadable] [unreadable] As the nervous system develops, compartment boundaries in the hindbrain become Wnt signaling centers and they induce new neurogenic zones in adjacent domains. Analysis of a Mind bomb interacting protein, Mosaic Eyes, showed that it is essential for the function of the genetic regulatory network that establishes and restricts Notch-dependent Wnt signaling centers to the boundaries. We have used computer simulations to visualize the dynamics of this genetic network. The simulations have helped understand how the signaling centers are established and how establishment of boundary-associated signaling centers could fail with dysfunction of specific components of the genetic network.[unreadable] Mib is an essential component of the Delta-Notch signaling system, which plays a critical role in determining how many progenitors are allowed to become neurons within a neurogenic domain. Mib ubiquitylates the transmembrane protein Delta at the cell surface and the ubiquitylated Delta is internalized from the surface. This step is essential for Delta to effectively activate its receptor, Notch, in neighboring cells. Mosaic Eyes (Moe) is a Mib-interacting protein that stabilizes Mib at the lateral surface of epithelial cells. While Moe is not essential during early neurogenesis, it is essential for regulating Notch function in the hindbrain, where Notch has a role in establishment of Wnt signaling centers at rhombomere boundaries. Reduction of Moe function results in failure of the mechanism that normally restricts Wnt signaling centers to boundaries and it allows spreading of the Wnt signaling center to adjacent non-boundary cells. In this context, Delta is expressed in para-boundary cells, where its interactions with Notch allow it to maintain Notch activation in adjacent boundary cells. At the same time, interactions of Delta with Notch within the para-boundary cells prevent Notch activation, and these interactions have a critical role in preventing Notch-mediated Wnt expression in non-boundary cells. Computer simulations of the genetic regulatory circuit that maintains Notch-dependent Wnt signaling at rhombomere boundaries reveal how loss of Delta-mediated inhibition of Notch signaling can account for failure to restrict Wnt signaling centers to rhombomere boundaries. Furthermore, the simulations illustrate how asymmetric signaling properties established in even and odd-numbered rhombomeres by early patterning mechanisms could set up the conditions for emergence of Wnt signaling centers at rhombomere boundaries later in development.